Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common cause of enterocolitis in humans and cattle but causes a systemic, typhoid-like, disease in susceptible mice. This facultative intracellular pathogen invades non-phagocytic cells, such as those found in the intestinal epithelium. Following internalization the bacteria are localized within a modified phagosome, known as the Salmonella-containing vacuoles (SCV). In epithelial cells, intracellular replication occurs within two distinct niches, the SCV and the cytosol, within which the bacterial growth rates and transcriptional activities differ. Interactions with epithelial cells are largely mediated by two type III secretion systems (T3SS1 and T3SS2), which translocate distinct cohorts of bacterial effector proteins into the host cell. Invasion is dependent on T3SS1 whereas T3SS2 is induced intracellularly and is associated with biogenesis of the SCV and intravacuolar survival/replication. Recently we have been investigating the relative importance of vacuolar versus cytosolic replication in epithelial cells. In approximately 5-20% of infected cells S. Typhimurium escape from the vacuole and survive and replicate in the cytosol. We have found that at later time points the T3SS1 is functionally active in the cytosolic bacteria whereas the T3SS2 is functionally active only in the vacuolar bacteria.